Indole compounds useful for the treatment of cancer

ABSTRACT

The present invention provides novel indole derivatives useful to inhibit cancer or sensitize cancer cells to chemotherapeutic agents, radiation or other anti-cancer treatments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/634,207 filed Aug. 9, 2000, which is a continuation-in-partof U.S. patent application Ser. No. 09/589,476, filed Jun. 7, 2000,which is a continuation-in-part of U.S. patent application Ser. No.09/360,020 filed Jul. 23, 1999, issued as U.S. Pat. No. 6,545,034, onApr. 8, 2003; which are incorporated by reference herein.

The invention was made with Government support under Grant No. 5ROIGM23200-24 awarded by the National Institute of Health. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Prostate cancer is the second leading cause of cancer death among malesin the United States. In 1998, an estimated 185,000 men were diagnosedwith prostate cancer, and more than 39,000 men died of the disease. See,S. H. Landis et al., Cancer Statistics, CA Cancer J. Clin., 48, 6(1998). Although survival rates are good for prostate cancer that isdiagnosed early, the treatments for advanced disease are limited tohormone ablation techniques and palliative care. Hormone ablationtechniques (orchiectomy and anti-androgen treatments) generally allowonly temporary remission of the disease. It usually recurs within 1–3years of treatment, with the recurrent tumors no longer requiringandrogens for growth and survival. D. G. Tang et al., Prostate, 32, 284(1997). Therapy with conventional chemotherapeutic agents, such asprogesterone, estramustine and vinblastine, has also not beendemonstrated to be effective to halt progression of the disease.

The number of nonsteroidal anti-inflammatory drugs (NSAIDs) hasincreased to the point where they warrant separate classification. Inaddition to aspirin, the NSAIDs available in the U.S. includemeclofenamate sodium, oxyphenbutazone, phenylbutazone, indomethacin,piroxicam, sulindac and tolmetin for the treatment of arthritis;mefenamic acid and zomepirac for analgesia; and ibuprofen, fenoprofenand naproxen for both analgesia and arthritis. Ibuprofen, mefenamic acidand naproxen are used also for the management of dysmenorrhea.

The clinical usefulness of NSAIDs is restricted by a number of adverseeffects. Phenylbutazone has been implicated in hepatic necrosis andgranulomatous hepatitis; and sulindac, indomethacin, ibuprofen andnaproxen with hepatitis and cholestatic hepatitis. Transient increasesin serum aminotransferases, especially alanine aminotransferase, havebeen reported. All of these drugs, including aspirin, inhibitcyclooxygenase, that in turn inhibits synthesis of prostaglandins, whichhelp regulate glomerular filtration and renal sodium and waterexcretion. Thus, the NSAIDs can cause fluid retention and decreasesodium excretion, followed by hyperkalemia, oliguria and anuria.Moreover, all of these drugs can cause peptic ulceration. See,Remington's Pharmaceutical Sciences, Mack Pub. Co., Easton, Pa. (18thed., 1990) at pages 1115–1122.

There is a large amount of literature on the effect of NSAIDs on cancer,particularly colon cancer. For example, see H. A. Weiss et al., Scand J.Gastroent., 31, 137 (1996) (suppl. 220) and Shiff et al., Exp. CellRes., 222, 179 (1996). More recently, B. Bellosillo et al., Blood, 92,1406 (1998) reported that aspirin and salicylate reduced the viabilityof B-cell CLL cells in vitro, but that indomethacin, ketoralac andNS-398, did not.

C. P. Duffy et al., Eur. J. Cancer, 34, 1250 (1998), reported that thecytotoxicity of certain chemotherapeutic drugs was enhanced when theywere combined with certain non-steroidal anti-inflammatory agents. Theeffects observed against human lung cancer cells and human leukemiacells were highly specific and not predictable; i.e., some combinationsof NSAID and agent were effective and some were not. The only conclusiondrawn was that the effect was not due to the cyclooxygenase inhibitoryactivity of the NSAID.

The Duffy group filed a PCT application (WO98/18490) on Oct. 24, 1997,directed to a combination of a “substrate for MRP”, which can be ananti-cancer drug, and a NSAID that increases the potency of theanti-cancer drug. NSAIDs recited by the claims are acemetacin,indomethacin, sulindac, sulindac sulfide, sulindac sulfone, tolmetin andzomepirac. Naproxen and piroxicam were reported to be inactive.

Recently, W. J. Wechter et al., Cancer Res., 60, 2203 (2000) reportedthat the NSAID, R-flurbiprofen, inhibited progression of prostate cancerin the TRAMP mouse, a prostate cancer model. The Wechter group filed aPCT application (WO98/09603) on Sep. 8, 1997, disclosing that prostatecancer can be treated with R-NSAIDs, including R(−)-etodolac andR-flurbiprofen. In contrast to R(−)-etodolac, the R-enantiomer offlurbiprofen and other (R)-2-aryl propionate NSAIDs are converted in thebody to the anti-inflammatory S-enantiomers, and hence are pro-drugs ofthe NSAIDs, while R(−)-etodolac is not per se an NSAID. Therefore, acontinuing need exists for effective methods to employ these preliminaryfindings to develop new compounds to treat neoplastic disease, includingprostate cancer and other cancers.

SUMMARY OF THE INVENTION

The present invention provides indole compounds of formula (I):

wherein R¹ is lower alkyl, lower alkenyl, (hydroxy)lower alkyl, loweralkynyl, phenyl, benzyl or 2-thienyl, R², R³, R⁴ and R⁵ are the same ordifferent and are each hydrogen or lower alkyl; each R⁶ is individuallyhydrogen, lower alkyl, hydroxy, (hydroxy)lower alkyl, lower alkoxy,benzyloxy, lower alkanoyloxy, nitro or halo, n is 1–3, R⁷ is hydrogen,lower alkyl or lower alkenyl, X is oxy and thio, Y is carbonyl,(CH₂)₁₋₃, (CH₂)₁₋₃C(O), or (CH₂)₁₋₃SO₂ and Z is (ω-(4-pyridyl)(C₂–C₄alkoxy), (ω-((R⁸)(R⁹)amino)(C₂–C₄ alkoxy), wherein R⁸ and R⁹ are each H,(C₁–C₃)alkyl or together with N are a 5- or 6-membered heterocyclic ringcomprising 1–3 N(R⁸), S or nonperoxide O; an amino acid ester of(ω-(HO)(C₂–C₄))alkoxy, N(R⁸)CH(R⁸)CO₂H, 1′-D-glucuronyloxy; Y-Z is(CH₂)₁₋₃R⁸ wherein R⁸ is OH, (C₂–C₄)acyloxy, SO₃H, PO₄H₂, N(NO)(OH),SO₂NH₂, PO(OH)NH₂, or tetrazolyl; or a pharmaceutically acceptable saltthereof.

The present invention also provides a therapeutic method to inhibit thegrowth of cancer cells and/or to sensitize cancer cells to inhibition bya chemotherapeutic agent. The method comprises contacting cancer cellswith an effective amount of the compound of formula (I), preferably incombination with a pharmaceutically acceptable carrier. The presentcompounds can be used to treat a mammal afflicted with cancer, such as ahuman cancer patient, and are preferably administered in conjunctionwith a chemotherapeutic agent, such as an alkylating agent or ananti-androgen, radiation and/or other anti-cancer therapy.

The present compounds are effective against hematopoietic cancers, suchas leukemias and cancers of the bone marrow, including chroniclymphocytic leukemia (CLL) and multiple myeloma (MM). The presentcompounds were unexpectedly found to be effective against cancer cellsthat express high levels of the nuclear hormone receptor, peroxisomeproliferator activated receptor-γ, (PPAR-γ), and/or high levels of theanti-apoptotic proteins, Mcl-1 and/or Bag-1. Such cancer cells includeat least some types of prostate cancer cells.

Activated PPAR-γ binds co-activator protein (CBP), a co-activator of theandrogen receptor known to be overexpressed in hormone-resistantprostate cancer.

Thus, compounds of formula (I) that activate PPAR-γ production canreduce the level of expression of the androgen receptor known to beover-expressed in hormone-resistant prostate cancer. Therefore, thepresent compounds can enhance the efficacy of conventional anti-androgentherapy, and can act to inhibit the spread of prostate cancer. Thecancer cells would be susceptible to inhibition by a compound of formula(I) when the level of PPAR-γ in the cells is sufficiently high, i.e.,the level is at least about fifty percent higher than the level ofPPAR-γ in normal prostate cells, as measured by a standard techniquesuch as, for example, immunoprecipitation or imunoblotting.

The present invention is based on the discovery by the inventors thatracemic etodolac inhibits the viability of purified CLL or MM cells atconcentrations that do not inhibit the viability of normal peripheralblood lymphocytes (PBLs). It was then unexpectedly found that the R(−)enantiomer of etodolac is as toxic to CLL cells as is the S(+)enantiomer. It was then found that etodolac synergistically interactedwith fludarabine and 2-chloroadenosine to kill CLL cells atconcentration at which the chemotherapeutic agents alone were inactive.Finally, it was found that both

R(−)- and S(+)-etodolac inhibited a number of prostate cancer celllines. Again the R(−) enantiomer was at least as effective as theS(+)-“anti-inflammatory” enantiomer. This was unexpected since the R(−)enantiomer of etodolac does not possess significant anti-inflammatoryactivity and is not converted to the S(+) enantiomer to a significantextent in vivo. As noted above, the R-enantiomers of otherR-2-arylpropionate NSAIDs are converted to the “active”anti-inflammatory S-enantiomers in vivo, and so function as pro-drugsfor the NSAID.

The extent of inhibition was markedly related to the level of expressionof PPAR-γ by the cell line. Cell lines with an elevated level of PPAR-γexpression were inhibited much more effectively than cell linesexpressing relatively low levels of PPAR-γ, as disclosed in the workingexamples.

A compound of formula (I) is preferred for practice of the presenttherapeutic method that does not exhibit undesirable bioactivities dueto inhibition of cyclooxygenase (COX) that are exhibited by some NSAIDs.However, the preferred compounds of formula (I) would not be consideredNSAIDs by the art, as they would not exhibit significantanti-inflammatory activity.

Thus, the present invention also provides a method for determiningwhether or not a particular cancer patient, such as a prostate cancerpatient, is amenable to treatment by a compound of formula (I),comprising isolating cancer cells and evaluating in vitro the relativelevel of PPAR-γ and/or Mcl-1 and/or Bag-1 relative to the level in acancer cell line, such as prostate cancer cell line, known to besusceptible to treatment by a compound of formula (I).

The present invention also provides a method to determine the ability ofa test agent to inhibit cancer cells, such as prostate cancer cells,comprising contacting a population of cancer cells, as from a prostatecancer cell line, with said agent and determining whether the agentincreases expression of PPAR-γ, or decreases the expression of Mcl-1and/or Bag-1 (or does both). The present invention also provides ageneral multilevel screening method to evaluate etodolac analogs, otherNSAIDs or other agents for their ability to inhibit cancer, preferablyetodolac-sensitive cancers, such as prostate cancer, CLL and MM. Agentsthat exhibit a positive activity, preferably at least equal to that ofR(−)-etodolac, or do not exhibit a negative activity, e.g., are no moreactive than R(−)-etodolac, are passed to the next screen.

Test agents are first evaluated for their ability to competitivelyinhibit the binding of etodolac, e.g., radiolabeled R(−)-etodolac to itsreceptor(s) on etodolac-sensitive cancer cells such as CLL cells. Agentsthat can compete effectively with R(−)-etodolac for etodolac bindingsite(s) on the cells are then evaluated in an assay to determine if theycan increase Ca⁺² uptake in cancer cells, such as CLL cells, preferablyas effectively as R(−)-etodolac. Agents that can induce intracellularCa⁺² uptake are screened to determine if they can induce chemokineticactivity (chemokinesis or chemotaxis) in a population of lymphocytes,such as B-CLL lymphocytes, preferably as effectively as R(−)-etodolac.Agents that are positive in this screen are then evaluated to determineif they can induce apoptosis or pro-apoptotic factors, such as increasedcaspase activity in cancer cells, such as CLL cells and other cancercells known to be etodolac sensitive, at least as effectively asR(−)-etodolac.

Agents that test positive in this screen are evaluated for their abilityto deplete lymphocytes in mice, and those that are no more active thanR(−)-etodolac are then evaluated in animal models of cancer to see ifthey can inhibit the induction of, or spread of cancer.

As used herein with respect to cancer or cancer cells, the term“inhibition” or “inhibit” includes both the reduction in cellularproliferation, blockage of cellular proliferation, or killing some orall of said cells. Thus, the term can be used in both the context of aprophylactic treatment to prevent development of cancer or as atreatment that will block, or slow the spread of established cancer.Whether or not the level of expression of a marker of susceptibility toetodolac treatment is sufficiently elevated to continue treatment withetodolac or an analog thereof is determined by comparison between therelative levels of expression of said marker in resistant andsusceptible cancer cell lines, as disclosed hereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the sensitivity of normal peripheral bloodlymphocytes (PBL) to racemic etodolac.

FIG. 2 is a graph depicting the sensitivity of CLL cells to racemicetodolac.

FIG. 3 is a graph depicting the synergistic effect of a combination ofracemic etodolac and fludarabine against CLL cells.

FIG. 4 is a graph depicting the synergistic effect of a combination of50 μM etodolac with 10 μM 2CdA or 10 mM Fludara against CLL cells.

FIG. 5 is a graph depicting the sensitivity of CLL cells to S- andR-etodolac.

FIGS. 6 and 7 depict the viability of CLL cells from two patients beforeand after etodolac administration.

FIGS. 8A–8D depict a flow cytometric analysis of CLL cells before andafter etodolac treatment.

FIGS. 9 and 10 depict the selective action of R(−)-etodolac against MMcells from two patients.

FIGS. 11A and 11B are a copies of a SDS-PAGE gels demonstrating thatetodolac induces a rapid downregulation in Mcl-1 (Panel A) and Bag-1(Panel B), that is blocked by MG-132.

FIG. 12 is a photocopy of an SDS-PAGE gel depicting expression of PPAR-γby seven cancer cell lines.

FIG. 13 is a graph depicting induction of PPAR-γ expression by etodolacand indomethacin.

FIG. 14 is a graph depicting expression of CD36 induced by etodolac andTGZ, in the presence and absence of TPA in human monocytes.

FIGS. 15A–15D are a copies of sections of prostate cancer tissue,untreated (A) or treated (B, C, D) with etodolac.

FIG. 16 is a graph depicting the detection of viable, apoptotic, anddead cells by flow cytometry using DiOC₆ and PI staining.

DETAILED DESCRIPTION OF THE INVENTION

Indole compounds of the present inventions include compounds of formula(I):

wherein R¹ is lower alkyl, lower alkenyl, (hydroxy)lower alkyl, loweralkynyl, phenyl, benzyl or 2-thienyl; R², R³, R⁴ and R⁵ are the same ordifferent and are each hydrogen or lower alkyl; each R⁶ is individuallyhydrogen, lower alkyl, hydroxy, (hydroxy)lower alkyl, lower alkoxy,benzyloxy, lower alkanoyloxy, nitro or halo, n is 1–3; R⁷ is hydrogen,lower alkyl or lower alkenyl; X is oxy or thio; Y is (CH₂)₁₋₃, or(CH₂)₁₋₃SO₂, and Z is (ω-(4-pyridyl)(C₂–C₄alkoxy),(ω-((R⁸)(R⁹)amino)(C₂–C₄ alkoxy), an amino acid ester of(ω-(HO)(C₂–C₄))alkoxy, N(R⁸)CH(R⁸)CO₂H, 1′-D-glucuronyloxy, orOCH₂CH₂N(CH₃)₃ ⁺; wherein R⁸ and R⁹ are each H, (C₁–C₃)alkyl or togetherwith N, are a 5- or 6-membered heterocyclic ring having 1–3 N(R⁸), S ornonperoxide O; or Y-Z is (CH₂)₁₋₃R¹⁰ wherein R¹⁰ is OH, (C₂–C₄)acyloxy,SO₃H, PO₄H₂, N(NO)(OH), SO₂NH₂, PO(OH)NH₂, provided that when n is 1, R⁶is hydrogen, and R¹ is methyl, then-Y-Z is not —CH₂CH₂—OH, —CH₂—OH,—CH₂CH₂—OC(O)CH₃, or —CH₂—OC(O)CH₃; or tetrazolyl; provided that when nis 1, R⁶ is 8-ethyl, and R¹ is ethyl, then —Y-Z is not —CH₂CH₂—OH, or—CH₂CH₂—OC(O)CH₃; or a pharmaceutically acceptable salt thereof.

As discussed above, the relatively low water solubility of the R(−)enantiomer of etodolac can reduce its usefulness against cancer whenadministered orally, or in an aqueous vehicle. Therefore, the presentinvention also provides novel indole compounds that exhibit enhancedwater solubility and/or bioavailability over the free acid or the simplealkyl esters of etodolac. Such analogs include (pyridinyl) lower alkylesters, (amino)lower alkyl esters, (hydroxy)lower alkyl esters and1′-D-glucuronate esters of etodolac, e.g., compounds of formula (II)wherein (a) Y is carbonyl and (b) Z is ((ω-(4-pyridyl)(C₂–C₄ alkoxy),(ω-((R⁸)(R⁹)amino)(C₂–C₄ alkoxy), wherein R⁸ and R⁹ are each H, (C_(1–C)₃)alkyl or together with N are a 5- or 6-membered heterocyclic ringcomprising 1–3 N(R⁸), S or nonperoxide O; an amino acid ester of(ω-(HO)(C₂–C₄)alkoxy, e.g., the L-valine or L-glycine ester of2-hydroxyethoxy, 1′-D-glucuronyloxy; and the pharmaceutically acceptablesalts thereof, e.g., with organic or inorganic acids. Other analogs ofincreased water solubility include amino acid amides, where Y iscarbonyl and Z is N(R⁸)CH(R⁸)CO₂H, and the pharmaceutically acceptablesalts thereof.

Such compounds can be prepared as disclosed in U.S. Pat. No. 3,843,681,U.S. patent application Ser. No. 09/313,048, Ger. Pat. No. 2,226,340(Amer. Home Products), R. R. Martel et al., Can. J. Pharmacol., 54, 245(1976); Demerson et al., J. Med. Chem., 19, 391 (1976); PCT applicationSerial No. US/00/13410 and Rubin (U.S. Pat. No. 4,337,760).

The resolution of racemic compounds of formula (I) can be accomplishedusing conventional means, such as the formation of a diastereomeric saltwith a optically active resolving amine; see, for example,“Stereochemistry of Carbon Compounds,” by E. L. Eliel (McGraw Hill,1962); C. H. Lochmuller et al., J. Chromatog., 113, 283 (1975);“Enantiomers, Racemates and Resolutions,” by J. Jacques, A. Collet, andS. H. Wilen, (Wiley-Interscience, New York, 1981); and S. H. Wilen, A.Collet, and J. Jacques, Tetrahedron, 33, 2725 (1977). For example, theracemate has been resolved by fractional crystallization of RS-etodolacusing optically active 1-phenylethylamine and HPLC has been used todetermine racemic etodolac and enantiomeric ratios of etodolac and twohydroxylated metabolites in urine (U. Becker-Scharfenkamp et al., J.Chromatog., 621, 199 (1993)). B. M. Adger et al. (U.S. Pat. No.5,811,558), disclosed the resolution of etodolac using glutamine andN(C₁–C₄ alkyl)-glutamine salts.

Etodolac itself (1,8-diethyl-1,3,4,9-tetrahydro[3,4–6]indole-1-aceticacid) is a NSAID of the pyranocarboxylic acid class, that was developedin the early 1970s. Its structure is depicted as formula (II), below,wherein (*) denotes the chiral center. See also, The Merck Index, (11thed.), at page 608.

The pharmacokinetics of etodolac have been extensively reviewed by D. R.Brocks et al., Clin. Pharmacokinet., 26, 259 (1994). Etodolac ismarketed as the racemate. The absolute configurations of the enantiomerswere found to be S(+) and R(−), which is similar to that for most otherNSAIDs. However, Demerson et al., J. Med. Chem., 26, 1778 (1983) foundthat the S(+)-enantiomer of etodolac possessed almost all of theanti-inflammatory activity of the racemate, as measured by reduction inpaw volume of rats with adjuvant polyarthritis, and prostaglandinsynthetase inhibitory activity of the drug. No anti-inflammatoryactivity was discernible with the R(−) enantiomer, and it is notconverted significantly to the S(+) enantiomer in vivo. Hence,R(−)-etodolac is not a NSAID. However, as disclosed below, R(−)-etodolacparadoxically was found to have potent activity against cancer cellsthat is at least equivalent to that of the S(+) enantiomer.

Etodolac possesses several unique disposition features due to theirstereoselective pharmacokinetics. In plasma, after the administration ofRS-etodolac, the concentrations of the “inactive” R-enantiomer ofetodolac are about 10-fold higher than those of the active S-enantiomer,an observation that is novel among the chiral NSAIDs. See, D. R. Brockset al., Clin. Pharmacokinet., 26, 259 (1994). After a 200 mg dose in sixelderly patients, the maximum plasma concentration of the R-enantiomerwas about 33 μM. In contrast, the maximum concentration of theS-enantiomer was 5-fold lower. The typical dosage of the racemic mixtureof etodolac is 400 mg BID, and the drug has an elimination half-lifebetween 6–8 hours. Moreover, it is likely that the administration of thepurified R-enantiomer will not display the side effects associated withcyclooxygenase (COX) inhibitors, such as ulcers and renal insufficiency,and thus can be given at considerably higher dosages. Nonetheless, therelatively low solubility of R(−)-etodolac in water can impede attainingplasma levels in humans that can inhibit cancer cells, particularlyprostate cancer cells. However, the compounds of formula (I) can bedissolved in water and other aqueous carriers at substantially higherconcentrations than R(−)-etodolac.

The compounds of formula (I) can also be prepared in the form of theirpharmaceutically acceptable salts or their non-pharmaceuticallyacceptable salts. The non-pharmaceutically acceptable salts are usefulas intermediates for the preparation of pharmaceutically acceptablesalts. Pharmaceutically acceptable salts are salts that retain thedesired biological activity of the parent compound and do not impartundesired toxicological effects. Examples of such salts are (a) acidaddition salts formed with inorganic acids, for example hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid andthe like; and salts formed with organic acids such as, for example,acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid,fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid,benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamicacid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonicacid, naphthalenedisulfonic acid, polygalacturonic acid, and the like;and (b) salts formed from elemental anions such as chlorine, bromine,and iodine. Preferred carboxylic acid salts are those of hydrophilicamines, such as glucamine or N—(C₁–C₄)alkylglucamine (see, Adger et al.(U.S. Pat. No. 5,811,558)).

The magnitude of a prophylactic or therapeutic dose of a compound orcompounds of formula (I) in the acute or chronic management of cancer,i.e., prostate cancer, will vary with the type and/or stage of thecancer, the adjunct chemotherapeutic agent(s) or other anti-cancertherapy used, and the route of administration. The dose, and perhaps thedose frequency, will also vary according to the age, body weight,condition, and response of the individual patient. In general, the totaldaily dose range for a compound or compounds of formula (I), for theconditions described herein, is from about 50 mg to about 5000 mg, insingle or divided doses. Preferably, a daily dose range should be about100 mg to about 4000 mg, most preferably about 1000–3000 mg, in singleor divided doses, e.g., 750 mg every 6 hr of orally administeredcompound. This can achieve plasma levels of about 500–750 μM, which canbe effective to kill cancer cells. In managing the patient, the therapyshould be initiated at a lower dose and increased depending on thepatient's global response. It is further recommended that infants,children, patients over 65 years, and those with impaired renal orhepatic function initially receive lower doses, particularly of analogswhich retain COX inhibitory activity, and that they be titrated based onglobal response and blood level. It may be necessary to use dosagesoutside these ranges in some cases. Further, it is noted that theclinician or treating physician will know how and when to interrupt,adjust or terminate therapy in conjunction with individual patientresponse. The terms “an effective inhibitory or amount” or “an effectivesensitizing amount” are encompassed by the above-described dosageamounts and dose frequency schedule.

Any suitable route of administration may be employed for providing thepatient with an effective dosage of a compound of formula (I). Forexample, oral, rectal, parenteral (subcutaneous, intravenous,intramuscular), intrathecal, transdermal, and like forms ofadministration may be employed. Dosage forms include tablets, troches,dispersions, suspensions, solutions, capsules, patches, and the like.The compound may be administered prior to, concurrently with, or afteradministration of chemotherapy, or continuously, i.e., in daily doses,during all or part of, a chemotherapy regimen. The compound, in somecases, may be combined with the same carrier or vehicle used to deliverthe anti-cancer chemotherapeutic agent.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrated agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. Tablets, capsules,pills, granules, microparticles and the like can also comprise anenteric coating, such as a coating of one of the Eudragit® polymers,that will permit release of the active compound(s) in the intestines,not in the acidic environment of the stomach. This can be advantageousin the case of elderly or frail cancer patients treated with anycompound that retains a significant COX-inhibitory activity, andconcomitant ulceration.

A syrup or elixir may contain the active compound, sucrose or fructoseas a sweetening agent, methyl and propylparabens as preservatives, a dyeand flavoring such as cherry or orange flavor. Of course, any materialused in preparing any unit dosage form should be pharmaceuticallyacceptable and substantially non-toxic in the amounts employed. Inaddition, the active compound may be incorporated into sustained-releasepreparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anon-toxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, non-toxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

Useful dosages of the compounds of formula I can be determined bycomparing their in vitro activity, and in vivo activity in animalmodels. Methods for the extrapolation of effective dosages in mice, andother animals, to humans are known to the art; for example, see U.S.Pat. No. 4,938,949.

Due to the ability of compounds of formula (I) that elevate PPAR-γlevels, to lower the expression of the androgen receptor known to beoverexpressed in hormone-refractory prostate cancer, compounds thatupregulate PPAR-γ are advantageously used in combination with steroidaland non-steroidal anti-androgens used in the treatment of prostatecancer. These compounds include leuprolide or goserelin acetate,bicalutamide and flutamide, nilutamide, cycloproterone acetate, amongothers.

Due to the ability of compounds of formula (I) that reduce PPAR-γ levelsto sensitize prostate cancer cells to killing by conventionalchemotherapeutic agents, such compounds can be employed withchemotherapeutic agents used to treat cancers such as prostate cancer,including estramustine, vinblastine, mitoxanthrone, prednisone and thelike, or melphalan to treat MM. Other chemotherapeutic agents,irradiation or other anti-cancer agents such as anti-tumor antibodies,or cytokines can be used with the present compounds. See, e.g.,Remington's Pharmaceutical Sciences (18th ed. 1990) at pages 1138–1162.

The invention will be further described by reference to the followingdetailed examples.

Preparation of Compounds of the Invention

General Chemistry. Pharmaceutical-grade tablets of racemic etodolac werepurchased from Watson Laboratories, Corona, Calif. All other reagentsand solvents were acquired from Aldrich, Milwaukee, Wis. Uncorrectedmelting points were determined on a Laboratory Device Mel-Temp IIcapillary melting point apparatus. Proton nuclear magnetic resonancespectra were recorded on a Varian Unity 500 NMR spectrophotometer at499.8 MHz or on a Varian Mercury NMR spectrophotometer at 400.06 MHz.The chemical shifts were reported in ppm on the δ scale from theindicated reference. Positive and negative ion loop mass spectra wereperformed by HT Laboratories, San Diego, Calif. Elemental analyses wereperformed by NuMega Resonance Labs, San Diego, Calif. Columnchromatography was conducted on E Merck silica gel (230–400 mesh) withthe indicated solvent system. Analytical thin layer chromatography (TLC)was conducted on silica gel 60 F-254 plates (EM Reagents).

EXAMPLE 12-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-ethanol (1)

A solution of etodolac (2.0 g, 6.97 mmol) in dry THF (5 mL) was addeddropwise to 1M LiAlH₄ in THF (10.5 mL, 10.5 mmol, 1.5 eq) over fiveminutes and stirred at room temperature overnight under argon. Theresulting mixture was then slowly quenched with EtOAc and poured overwater to form an emulsion. The emulsion was filtered, and the aqueouslayer was separated and extracted twice with EtOAc. The three organicphases were combined, washed with brine, dried with Na₂SO₄,concentrated, and purified by column chromatography using50:50:EtOAc:Hexane to give a yellow oil (1.87 g, 98%): ¹HNMR (CDCl₃, δTMS): 0.95 (t, 3H), 1.36 (t, 3H), 1.96 (m, 2H), 2.14 (m, 2H), 2.68 (br,OH), 2.81 (m, 2H), 2.83 (q, 2H), 3.70 (m, 2H), 4.07 (m, 2H), 7.02–7.39(m, 3H, Ar—H), 7.74 (br, 1H, NH). MS⁺: m/z 296 (MNa⁺). MS⁻: m/z 308(MCl⁻), 272 ([M−H]⁻).

EXAMPLE 21,8-Diethyl-1-(2-methoxyethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (2)

To a solution of compound 1 (348 mg, 1.27 mmol) in dry THF (5 mL) underargon, 60% NaH in mineral oil (64 mg, 1.59 mmol, 1.25 eq) was added in aportion-wise manner. After stirring for thirty minutes, MeI (99 μL, 1.59mmol, 1.25 eq) was added dropwise, and the reaction was stirred for 2days at room temperature. The resulting mixture was diluted with brineand extracted three times with Et₂O. The combined organic phases weredried with Na₂SO₄, concentrated, and purified by column chromatographyusing 20:80:EtOAc:Hexane to give a yellow-white solid (228 mg, 62%): mp125–126° C. ¹HNMR (CDCl₃, δ TMS): 0.86 (t, 3H), 1.37 (t, 3H), 1.94 (m,2H), 2.15 (m, 2H), 2.78 (t, 2H), 2.85 (q, 2H) 3.37 (s, 3H), 3.52 (m,2H), 4.00 (m, 2H), 6.99–7.38 (m, 3H, Ar—H), 8.43 (br, 1H, NH). MS⁻: m/z286 ([M−H]⁻). Anal. (C₁₈H₂₅NO₂): C, H, N.

EXAMPLE 31,8-Diethyl-1-(2-fluoroethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole (3)

To a stirred solution of compound 1 (136 mg, 0.5 mmol) in CH₂Cl₂ (2 mL)at −40° C. under argon, DAST (396 μL, 3.0 mmol, 6.0 eq) was slowly addedin a dropwise manner. The resulting mixture was allowed to warm to roomtemperature and stirred for one hour before being cooled to 0° C. andquenched with MeOH (1 mL). The mixture was stirred an additional thirtyminutes at room temperature, and then saturated NaHCO₃ (10 mL) was addeddropwise. The resulting aqueous layer was extracted three times withCH₂Cl₂. The combined organic phases were dried with Na₂SO₄,concentrated, and purified by column chromatography using5:95:EtOAc:Hexane to give a yellow-white solid (45 mg, 33%): mp 118–119°C. ¹HNMR (CDCl₃, δ TMS): 0.88 (t, 3H), 1.37 (t, 3H), 1.94 (m, 2H), 2.29(m, 2H), 2.78 (m, 2H), 2.86 (q, 2H), 3.99 (m, 2H), 4.57 (qq, 2H),7.03–7.38 (m, 3H, Ar—H), 7.65 (br, 1H, NH). MS⁻: m/z 310 (MCl⁻), 274([M−H]⁻). Anal. (C₁₇H₂₂NOF): C, H, N.

EXAMPLE 41-(2-Chloroethyl)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (4)

A solution of compound 1 (136 mg, 0.5 mmol), PPh₃ (262 mg, 1.0 mmol, 2.0eq), and CCl₄ (2.5 mL) was refluxed overnight. The resulting mixture wasthen diluted with water and extracted three times with CH₂Cl₂. Theorganic phases were combined, dried with Na₂SO₄, concentrated, andpurified by column chromatography using 5:95:EtOAc:Hexane to give awhite solid (74 mg, 51%): mp 106–107° C. (dec). ¹HNMR (CDCl₃, δ TMS):0.93 (t, 3H), 1.37 (t, 3H), 1.91 (m, 2H), 2.34 (m, 2H), 2.76 (m, 2H),2.87 (q, 2H), 3.46 (dm, 2H), 3.99 (m, 2H), 7.04–7.38 (m, 3H, Ar—H), 7.55(br, 1H, NH). MS⁻: m/z 326 (MCl⁻), 290 ([M−H]⁻). Anal. (C₁₇H₂₂NOCl): C,H, N.

EXAMPLE 5 1,1,8-Triethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole (5)

To a stirred solution of compound 4 (103 mg, 0.35 mmol), AlBN (11 mg,0.07 mmol, 0.2 eq), and toluene (3 mL) at room temperature under argon,HSnBu₃ (380 μL, 1.41 mmol, 4.0 eq) was added in a dropwise manner. Theresulting mixture was stirred overnight at 110° C. and concentrated. Theresidue was diluted with hexane and extracted three times withacetonitrile. The acetonitrile layers were combined, concentrated, andpurified by column chromatography using 3:97:EtOAc:Hexane to give anoff-white solid (56 mg, 62%): mp 133–134° C. ¹HNMR (CDCl₃, δ TMS): 0.88(t, 6H), 1.37 (t, 3H), 1.87 (q, 4H), 2.78 (t, 2H), 2.86 (q, 2H), 4.02(t, 2H), 7.01–7.38 (m, 3H, Ar—H), 7.46 (br, 1H, NH). MS⁻: m/z 256([M−H]⁻). Anal. (C₁₇H₂₃NO): C, H, N.

EXAMPLE 62-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-acetamide (6)

To a stirred solution of etodolac (287 mg, 1.0 mmol) in CH₂Cl₂ (5 mL) atroom temperature, oxalyl chloride (105 μL, 1.2 mmol, 1.2 eq) was addeddropwise, followed by a catalytic amount of DMF (2 drops). The mixturewas stirred for one hour and then concentrated. The resulting orangesolid was dissolved in dry THF (2 mL) and added dropwise to a stirredsolution of ice-cold concentrated NH₄OH (5 mL). The mixture was allowedto warm to room temperature, stirred for two days, diluted with brine,and extracted three times with EtOAc. The combined organic phases weredried with Na₂SO₄, concentrated, and purified by column chromatographyusing 50:50:EtOAc:Hexane to give a yellow-white solid (100 mg, 35%): mp189–190° C. ¹HNMR (CDCl₃, 67 TMS): 0.87 (t, 3H), 1.32 (t, 3H), 2.08 (m,2H), 2.83 (m, 4H), 2.91 (q, 2H), 4.07 (m, 2H), 5.48 (br, 1H, CONH₂),6.33 (br, 1H, CONH₂), 6.99–7.36 (m, 3H, Ar—H), 9.26 (br, 1H, NH). MS⁺:m/z 309 (MNa⁺). MS⁻: m/z 321 (MCl⁻), 286 (M⁻). Anal.(C₁₇H₂₂N₂O₂.0.125H₂O): C, H, N.

EXAMPLE 72-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-yl)-ethylideneamine(7)

A solution of compound 6 (119 mg, 0.42 mmol) in dry THF (3 mL) was addeddropwise to 1M LiAlH₄ in THF (624 μL, 0.62 mmol, 1.5 eq) and stirred fortwo hours at room temperature under argon. Another equivalent of 1MLiAlH₄ in THF (420 μL, 0.42 mmol, 1.0 eq) was then added and thereaction was stirred overnight. The resulting mixture was then slowlyquenched with EtOAc and poured over water to form an emulsion. Theemulsion was filtered, and the aqueous layer was separated and extractedtwice with EtOAc. The three organic phases were then combined, driedwith Na₂SO₄, concentrated, and purified by column chromatography using50:50:EtOAc:Hexane to give a yellow-brown solid (40 mg, 36%): mp101–103° C. ¹HNMR (CDCl₃, δ TMS): 0.94 (t, 3H), 1.38 (t, 3H), 1.96 (m,2H), 2.73 (qd, 2H), 2.87 (q, 2H), 2.99 (t, 2H), 3.32 (m, 1H, CH═N), 3.87(t, 2H), 7.04–7.43 (m, 3H, Ar—H), 7.89 (br, 1H, NH). MS⁺: m/z 293(MNa⁺), 271 (MH⁺). MS⁻: m/z 269 ([M−H]⁻). Anal. (C₁₇H₂₂N₂O.0.5H₂O): C,H, N.

EXAMPLE 8(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-acetaldehyde (8)and1,8-Diethyl-1-(2-methylsufanylmethoxyethyl)-1,3,4,9-tetrahydropyrano[3,4-b]indole(9)

A solution of compound 1 (136 mg, 0.5 mmol), dry DMSO (1.5 mL), and dryAc₂O (1.0 mL) was stirred overnight at room temperature. The reactionmixture was then diluted with water and extracted three times with Et₂O.The combined organic phases were washed with saturated NaHCO₃, driedwith Na₂SO₄, concentrated, and purified by column chromatography using10:90:EtOAc:Hexane to give compound 8 (yellow-white solid, 72 mg, 53%)and compound 9 (yellow oil, 60 mg, 36%).

For compound 8, mp 123–124° C. ¹HNMR (CDCl₃, δ TMS): 0.87 (t, 3H), 1.37(t, 3H), 2.04 (m, 2H), 2.81 (m, 2H), 2.87 (q, 2H), 3.07 (s, 2H), 4.02(m, 2H), 7.03–7.38 (m, 3H, Ar—H), 8.36 (br, 1H, NH), 9.78 (s, 1H, CHO).MS⁻: m/z 270 ([M−H]⁻). TLC (20:80:EtOAc:Hexane): Rf(7)=0.37. Anal.(C₁₇H₂₁NO₂.0.1C₄H₈O₂.0.1C₆H₁₄.0.75H₂O): C, H, N.

For compound 9, ¹HNMR (CDCl₃, δ TMS): 0.88 (t, 3H), 1.36 (t, 3H), 1.94(m, 2H), 2.14 (s, 3H), 2.20 (m, 2H), 2.79 (t, 2H), 2.86 (q, 2H), 3.66(dq, 2H), 4.06 (q, 2H), 4.62 (s, 2H), 7.01–7.38 (m, 3H, Ar—H), 8.21 (br,1H, NH). MS⁻: m/z 332 ([M−H]⁻). TLC (20:80:EtOAc:Hexane): Rf (8)=0.46.Anal. (C₁₉H₂₇NO₂S.0.5H₂O): C, H, N.

When the reaction mixture is allowed to stir overnight at 50° C.compound 9 was afforded as the major product.

EXAMPLE 9 Alternative Synthetic Method for(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-acetaldehyde (8)

To a stirred solution of compound 1 (136 mg, 0.5 mmol), CH₂Cl₂ (2 mL),dry DMSO (142 μL, 2.0 mmol, 4.0 eq), and dry TEA (697 μL, 5.0 mmol, 10.0eq) at room temperature under argon, sulfur trioxide-pyridine complex(Pyr-SO₃, 318 mg, 2.0 mmol, 4.0 eq) was added in a portion-wise mannerand stirred for two days. The resulting mixture was then diluted withwater and extracted three times with Et₂O. The combined organic phaseswere dried with Na₂SO₄, concentrated, and purified by columnchromatography using 20:80:EtOAc:Hexane to give a yellow-white solid (70mg, 52%). Spectral data were identical to those reported above.

EXAMPLE 10 AceticAcid-2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-ethylester (10)

A solution of compound 1 (273 mg, 1.0 mmol), dry DMSO (3 mL), dry Ac₂O(2 mL), and dry TEA (1 mL) was stirred overnight at room temperature.The reaction mixture was then diluted with water and extracted threetimes with EtOAc. The combined organic phases were washed with saturatedNaHCO₃ and brine, dried with Na₂SO₄, concentrated, and purified bycolumn chromatography using 10:90:EtOAc:Hexane to give a yellow solid(245 mg, 78%): mp 126–127° C. ¹HNMR (CDCl₃, δ TMS): 0.91 (t, 3H), 1.38(t, 3H), 1.88 (s, 3H), 1.90 (m, 2H), 2.21 (t, 2H), 2.78 (m, 2H), 2.87(q, 2H), 4.01 (t, 2H), 4.14 (m, 2H), 7.02–7.37 (m 3H, Ar—H), 7.71 (br,1H, NH). MS⁻: m/z 350 (MCl⁻), 314 ([M−H]⁻). Anal. (C₁₉H₂₅NO₃): C, H, N.

EXAMPLE 112-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-ethane-1,1-diol(11)

Dry DMSO (71 μL, 1.0 mmol, 2.0 eq) was added dropwise to a solution ofoxalyl chloride (66 μL, 0.75 mmol, 1.5 eq) in CH₂Cl₂ (1 mL) at −78° C.After stirring for ten minutes, a solution of compound 1 (136 μL, 0.5mmol) in CH₂Cl₂ (4 mL) was added dropwise, and the reaction was stirredfor an additional thirty minutes at −78° C. before the addition of TEA(2.79 μL, 2.0 mmol, 4.0 eq). The reaction mixture was then allowed towarm slowly to 0° C., diluted with saturated NH₄Cl, and extracted twicewith Et₂O. The combined organic phases were washed with water and brine,dried with Na₂SO₄, concentrated, and purified by column chromatographyusing a gradient of 50:50:EtOAc:Hexane to 100% EtOAc to give a whitesolid (46 mg, 32%): mp 158–159° C. ¹HNMR (CDCl₃, δ TMS): 0.50 (t, 3H),1.25 (t, 3H), 1.50 (m, 1H), 1.82 (m, 1H), 2.04 (m, 1H), 2.17 (m, 1H),2.33 (m, 1H), 2.58 (q, 2H), 2.88 (m, 2H), 3.77 (m, 1H), 3.91 (m, 1H),4.19 (q, 1H), 4.33 (m, 1H), 7.02–7.15 (m, 3H, Ar—H), 7.66 (br, 1H, NH).MS⁻: m/z 288 ([M−H]⁻). Anal. (C₁₇H₂₃NO₃): C, H, N.

EXAMPLE 121-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-propan-2-ol(12)

A solution of compound 8 (275 mg, 1.01 mmol) in dry THF (4 mL) was addeddropwise over five minutes to a stirred solution of 1M MeMgCl in THF(1.52 mL, 1.52 mmol, 1.5 eq) at 0° C. under argon and allowed to warmslowly to room temperature. After stirring for two hours, anotherequivalent of 3M MeMgCl in THF (337 μL, 1.01 mmol, 1.0 eq) was added,and the mixture was stirred for an additional hour. The mixture was thendiluted with saturated NaHCO₃ and extracted three times with EtOAc. Thecombined organic phases were dried with Na₂SO₄, concentrated, andpurified by column chromatography using 20:80:EtOAc:Hexane to give ayellow solid (280 mg, 96%): mp 103–104° C. ¹HNMR (CDCl₃, δ TMS) shows a2:1 mixture of diastereomers. MS⁻: m/z 322 (MCl⁻), 286 ([M−H]⁻). Anal.(C₁₈H₂₅NO₂): C, H, N. The product was carried on to the next step as amixture of diastereomers.

EXAMPLE 131-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-yl)-propan-2-one(13)

To a solution of compound 12 (77 mg, 0.27 mmol), CH₂Cl₂ (2 mL), dry DMSO(76 μL, 1.07 mmol, 4.0 eq), and dry TEA (374 μL, 2.68 mmol, 10.0 eq) atroom temperature under argon, sulfur trioxide-pyridine complex (Pyr-SO₃,171 mg, 1.07 mmol, 4.0 eq) was added in a portion-wise manner andstirred overnight. The resulting mixture was then diluted with brine andextracted three times with EtOAc. The combined organic phases were driedwith Na₂SO₄, concentrated, and purified by column chromatography using15:85:EtOAc:Hexane to give an off-white solid 39 mg, 51%): mp 154–155°C. ¹HNMR (CDCl₃, δ TMS): 0.80 (t, 3H), 1.37 (t, 3H), 2.02 (m, 2H), 2.21(s, 3H), 2.78 (m, 2H), 2.88 (q, 2H), 3.14 (s, 2H), 3.98 (m 2H),7.01–7.37 (m, 3H, Ar—H), 9.01 (br, 1H, NH). MS⁻: m/z 284 ([M−H]⁻). Anal.C₁₈H₂₃NO₂): C, H, N.

EXAMPLE 14 Isolation of Racemic Etodolac(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indole-1-acetic Acid)

Pharmaceutical-grade tablets of racemic etodolac (20×400 mg) werecrushed to a fine powder with a mortar and pestle. The powder was thenstirred in hot EtOAc (150 mL) for ten minutes and vacuum filteredthrough a Buchner funnel. This process was repeated two more times, andthe three filtrates were combined and concentrated to give an off-whitesolid (7.92 g, 99%): ¹HNMR (DMSO, δ TMS): 0.61 (t, 3H), 1.24 (t, 3H),2.03 (q, 2H), 2.63 (m, 2H), 2.82 (dd, 2H), 2.83 (q, 2H), 3.92 (m, 2H),6.86–7.23 (m, 3H, Ar—H), 10.47 (br, 1H, NH), 12.00 (br, COOH).

EXAMPLE 15 Sensitivity of Normal Peripheral Blood Lymphocytes and CLLCells to Etodolac

Mononuclear cells were isolated from the peripheral blood of B-CLLpatients and normal donors using density gradient centrifugation(Ficoll-Paque). Cells were cultured at 2×10⁶ cells per mL in RPMI with20% autologous plasma in 96-well plates with or without the indicated μMconcentrations of etodolac (racemic, S-etodolac, R-etodolac) and incombination with 2-chloro-2′-deoxyadenosine (2CdA) or fludarabine. Atindicated times (12, 24, 36, 48, 60, 72 hours), viability assays wereperformed using the erythrocin B exclusion assay, as described by D.Carson et al., PNAS USA, 89 2970 (1992).

As shown in FIG. 1, significant death of normal PBLs occurred only at800 μM racemic etodolac, a concentration which cannot be obtained invivo.

Peripheral blood lymphocytes from a normal donor were cultured with 1.0mM etodolac for 24 hours. Then B lymphocytes were identified by stainingwith anti-CD 19 antibody, and viability was assessed by DiOC₆fluorescence. Etodolac under these conditions did not reduce theviability of the normal B cells, compared to control cultures. When thesame viability assay was run with purified CLL cells from the peripheralblood of a CLL patient, the results were different. As shown in FIG. 2,50% of the CLL cells were killed by a 48 hour exposure to 200 μM racemicetodolac. More than 95% of the treated cells were malignant Blymphocytes.

EXAMPLE 16 Synergistic Combinations of Etodolac and ChemotherapeuticAgents

Fludarabine is a nucleoside analog commonly used for the treatment ofCLL. In this experiment the in vitro survival of CLL cells at theindicated time points was compared in cultures containing medium alone(“Con”, squares), fludarabine 10 nM (diamonds), etodolac 10 μM (closedcircles), and fludarabine 10 nM plus etodolac 10 μM (open circles). Thetwo drugs together exhibited a synergistic cytotoxic effect. FIG. 3shows that the combination killed 50% of CLL cells during 48 hours ofculture, while either drug alone was ineffective. FIG. 4 demonstratessynergy between 50 μM etodolac and 10 nM 2-chlorodeoxy-adenosine andfludarabine, under the same test conditions.

EXAMPLE 17 Effect of R(−) and S(+) Etodolac Against CLL Cells

Etodolac tablets were ground in a mortar and extracted from theformulation using ethyl acetate. The resulting racemic mixture ofenantiomers was separated into R and S isomers on a preparative scale byfractional crystallization by the procedure of Becker-Scharfenkamp andBlaschke, J. Chromatog., 621, 199 (1993). Thus, the racemic mixturesolid was dissolved in absolute 2-propanol and S-1-phenylethylamine wasadded to the solution. The resulting salt solution was stored in therefrigerator for 4 days. The crystalline white salt product was filteredand washed with cold 2-propanol and recrystallized two more times from2-propanol. The same procedure was repeated for the R isomer only usingR-1-phenylethylamine as the resolving agent. Finally, the R and S saltswere decomposed using 10% sulfuric acid (v/v) and extracted with ethylacetate. The chiral purity of each isomer was verified by HPLC using aChiral-AGP column from ChromTech.

The toxicities of the two enantiomers to CLL cells cultured in RPMI 1640medium with 10% autologous plasma were compared at the indicatedconcentrations and time points, as shown in FIG. 5. The R- andS-enantiomers are equivalently cytotoxic to the CLL cells.

EXAMPLE 18 Viability of CLL Cells Before and After Etodolac Treatment

Heparinized blood was taken from two patients (JK and NA) with CLL. Theneach patient immediately took a 400 mg etodolac tablet, and a secondtablet 12 hours later. After another 12 hours, a second blood specimenwas obtained. The CLL cells were isolated and their survival in vitrowere compared in RPMI 1640 medium containing 10% autologous plasma, asdescribed in Example 1. The circles show CLL cells before etodolactreatment. In FIGS. 6–7, the upward pointing triangles represent CLLcell viability after etodolac treatment, wherein the cells are dispersedin medium containing the pretreatment plasma. The downward pointingtriangles are CLL cells after treatment maintained in medium with thepost-treatment plasma.

FIG. 6 shows the different survivals of the two cell populations frompatient JK. Note that the cells after treatment had a shortened survivalcompared to the cells before treatment. FIG. 7 shows a less dramatic butsimilar effect with patient NA. FIG. 8 is a flow cytometric analysis ofCLL cells from patient JK before and after etodolac treatment. DiOC₆ isa dye that is captured by mitochondria. When cells die by apoptosis, theintensity of staining decreases. The X axis on the four panels in FIG. 8shows the DiOC₆ staining. An increased number of dots in the left lowerbox indicates cell death by apoptosis. If one compares the cells takenfrom the patient before etodolac treatment, and after etodolactreatment, one can see that the number of dots in the left lower box ismuch higher after the drug. This effect is detectable at 12 hours, andincreases further after 24 hours.

To conduct the flow cytometric analysis, the mitochondrial transmembranepotential was analyzed by 3,3′ dihexyloxacarboncyanide iodide (DiOC₆),cell membrane permeability by propidium iodide (PI)³ and mitochondrialrespiration by dihydrorhodamine 123 (DHR) (See J. A. Royall et al.,Arch. Biochem. Biophys., 302, 348 (1993)). After CLL cells were culturedfor 12 or 24 hours with the indicated amount of etodolac, the cells wereincubated for 10 minutes at 37° C. in culture medium containing 40 nM ofDiOC₆ and 5 μg/ml PI. Cells were also cultured for 3 hours with theindicated amount of etodolac, spun down at 200×g for 10 minutes andresuspended in fresh respiration buffer (250 mM sucrose, 1 g/L bovineserum albumin, 10 mM MgCl₂, 10 mM K/Hepes, 5 mM KH₂PO₄ (pH 7.4)) andcultured for 10 minutes at 37° C. with 0.04% digitonin. Then cells wereloaded for 5 minutes with 0.1 μM dihydrorhodamine (DHR). Cells wereanalyzed within 30 minutes in a Becton Dickinson FAC-Scaliburcytofluorometer. After suitable comprehension, fluorescence was recordedat different wavelength: DiOC₆ and DHR at 525 nm (Fl-1) and PI at 600 nm(FL-3).

As a general matter a reduction of 10% in the survival of thepost-treatment malignant cells, compared to the pretreatment malignantcells, at 16 hours after culture in vitro is considered a “positive” inthis test, and indicates the use of etodolac, i.e., R(−)-etodolac in CLLor other cancer therapy.

EXAMPLE 19 Ability of R(−)-Etodolac to Selectively Kill MM Cells

Bone marrow was obtained from two patients with multiple myeloma. Themarrow contained a mixture of malignant cells, as enumerated by highlevel expression of the CD38 membrane antigen, and normal cells. Thesuspended marrow cells were incubated for 72 hours in RPMI 1640 mediumwith 10% fetal bovine serum, and various concentrations of the purifiedR-enantiomer of etodolac. Then the dead cells were stained withpropidium iodide, and the multiple myeloma cells were stained withfluorescent monoclonal anti-CD38 antibodies. The data were analyzed byfluorescent activated cell sorting. FIGS. 9–10 show that R-etodolac didnot kill the normal bone marrow cells (light bars), but dose-dependentlykilled the multiple myeloma cells (dark shaded areas), in the marrowcells from both patients.

EXAMPLE 20 Etodolac Cytotoxicity to Cancer Cell Lines

Table 1 summarizes the cytotoxic effects of R(−)-etodolac towardprostate cancer cell lines and one colon cancer cell line are indeedwithin clinically achievable concentrations, given that a 1 gram dosageof R(−)-etodolac should yield a maximal plasma concentration in a humansubject of about 400 μM. The fact that the R(−)- and S(+)-enantiomersare both cytotoxic indicates that the anti-prostate cancer activity isCOX independent. Note that R(−)-etodolac, which is devoid ofanti-inflammatory activity, nonetheless is more toxic to prostate cancercells than is S(+) etodolac.

TABLE 1 Cell line Origin Etodolac R/S Etodolac R Etodolac S PhenotypePC-3 Prostate 340 ± 20* 150 ± 15* 800 + 30* Sensitive LNCaP- Prostate400 ± 35  270 ± 50  220 ± 20  Sensitive FGC Alva-31Prostate >1000 >1000 >1000 Resistant OVCAR-3 Ovarian >1000 >1000 >1000Resistant MDA- Breast >1000 >1000 >1000 Resistant MB-231 HCT-116 Colon450 ± 15  280 ± 20  420 ± 50  Sensitive SW260 Colon 1000 ± 120  ND NDResistant A549 Lung >1000 >1000 >1000 Resistant *IC₅₀ (μM) of EtodolacR/S, R or S. Cytotoxicity was assessed by MTT assay after three dayscontinuous exposure to decreasing concentrations of the agent. Theresults were confirmed by FACS using propidium iodide uptake.

EXAMPLE 21 Etodolac Downregulation of Mcl-1 and Bag-1

As planar hydrophobic compounds, etodolac and other NSAIDS can readilyinsert into cell and organ membranes, and can disrupt their structureand function (S. B. Abramson et al., Arthritis and Rheumatism, 32, 1(1989)). The proteins Mcl-1 and Bag-1 are anti-apoptotic members of thebcl-2 family that are found in mitochondria (X. Wang et al., Exp. CellRes., 235, 210 (1997)). As early as two hours after incubation with 100μM etodolac, Mcl-1 and Bag-1 levels fell in an etodolac sensitiveprostate cancer cell line (LNCaP). The fall in Mcl-1 and Bag-1 levelswas prevented by co-incubation of the prostate cells with 5.0 μM MG-132,a recently described inhibitor of the proteasome (FIG. 11, Panels A andB, respectively) (D. H. Lee at al., Trends Cell Biol., 8, 397 (1998)).Detergent lysates (20 μg per lane) were subjected to SDS-PAGE andimmunoblotted with anti-Mcl-1 and anti-Bag-1 antibodies. Pre-incubationof the cells with Z-VAD, a broad-spectrum caspase inhibitor, did notprevent the Mcl-1 and Bag-1 downregulation. Etodolac incubation did notalter Bcl-2 and Bax levels (data not shown). Thus, etodolac did notinterfere with Mcl-1 synthesis, but probably accelerated its turnover.Both R- and S-etodolac induced Mcl-1 degradation at equivalentconcentrations.

EXAMPLE 22 Expression of PPAR-γ in Cancer Cell Lines

Although etodolac has not been previously studied, high concentrationsof other NSAIDs have been reported to activate the nuclear hormonereceptor PPAR-γ (J. M. Lehmann et al., J. Biol. Chem., 272, 3406 (1997).Moreover, maximal activation of PPAR-γ induces apoptosis in humanmacrophages (G. Chinetti et al., J. Biol. Chem., 273, 25579 (1998).Therefore, it was of interest to determine if prostate cells expressPPAR-γ, and to compare the expression level with other cancer types.Detergent lysates (20 μg per lane) obtained from subconfluent cell lineswere subjected to SDS-PAGE and immunoblotted with anti-PPAR-γantibodies. To normalize the PPAR-γ content, the membrane was reblottedwith an anti-actin monoclonal antibody. Lane 1: PC-3, Lane 2: SW260,Lane 3: A549, Lane 4: MDA-MB-231, Lane 5: Alva-31, Lane 6: LNCaP, Lane7: HCT-116 (see Table 1). It was observed that some etodolac-susceptibleprostate cells (PC3 especially) expressed remarkably high levels ofimmunoreactive PPAR-γ (FIG. 12).

EXAMPLE 23 Activation of PPAR-γ by Etodolac

RAW264.7 cells were transfected at a density of 3×10⁵ cells/ml in sixwell plates using lipofectamine with the PPAR-γ expression vectorpCMX-PPAR-γ (0.1 μg), and the PPAR-γ reporter construct (AOx)₃-TK-Luc (1μg) as previously described by M. Ricote et al., Nature, 391, 79 (1998).Cells were treated for 24 hours with the compounds indicated on FIG. 13,harvested and assayed for luciferase activity. Results are expressed asthe mean±SD. As shown in FIG. 13, both the R- and S-enantiomers ofetodolac activated a PPAR-γ reporter gene construct at concentrationsreadily achieved in human plasma after in vivo administration. THP-1human monocytic cells (ATCC) were incubated in the presence or absenceof phorbol ester (40 ng TPA) and 200 μM racemic etodolac or 20 μMtroglitazone. After three days of culture, the surface expression of thescavenger receptor CD36 was measured by flow cytometry. As shown in FIG.14, both R- and S-etodolac caused the expression of CD36, a marker ofPPAR-γ activation, in the human cell line THP-1 during macrophagedifferentiation.

EXAMPLE 24 Etodolac Treatment of Prostate Cancer Tissue Samples

Freshly obtained prostatectomy samples were cut into 3 mm³ pieces, andincubated for 72 hours in RPMI-1640 supplemented with 10% FBS andantibiotics in the absence (A, 400×) or presence of racemic etodolac (B,400×) or the purified R enantiomer (C, 400×; and D, 630×). The tissueswere next fixed in 4% paraformaldehyde in PBS, embedded in paraffin,sectioned and stained with hematoxylin and eosin. FIG. 15A shows theinfiltrating tumor cells (large nuclei) and some residual normalepithelium. FIGS. 15B to 15D show the effect of etodolac: note theabundant presence of pyknotic apoptotic nuclei (dark arrows, B and D),and the disintegration of the neoplastic glandular architecture (B+C).Etodolac was found to be selectively toxic to the tumor cells, but didnot affect normal basal cells. The racemic mixture (R/S) and thepurified R and S analogs were found both active.

EXAMPLE 25 Apoptotic Assays

The compounds of the invention were screened for CLL apoptotic activityby flow cytometry and a MTT-based assay. Primary CLL cells isolated frompatients were used in both studies. Primary CLL cells were kindlyprovided by Dr. Thomas Kipps, University of California at San Diego, LaJolla, Calif. Primary PBL cells were acquired from the San Diego BloodBank, San Diego, Calif. DiOC₆ and PI dyes were obtained from MolecularProbes, Eugene, Oreg. Otherwise, unless indicated, all other reagentswere purchased from Sigma (St. Louis, Mo.), and all test compounds weredissolved in sterile DMSO.

Flow Cytometry Studies.

In the flow cytometry experiment, the cells were incubated withindividual test compounds and stained with 3,3′-dihexyloxacarbocyanineiodide (DiOC₆), a cationic dye attracted to the mitochondrialtransmembrane potential, and propidium iodide (PI), amembrane-impermeable nucleic acid dye. Viable cells (DiOC₆ ⁺, PI⁻) withfunctional mitochondria and an intact cell membrane retained DiOC₆ andexcluded PI. In contrast, apoptotic cells (DiOC₆ ⁻, PI⁻) failed toabsorb DiOC₆ because of their reduced mitochondrial potential resultingfrom apoptosis. The results were determined using the techniques ofZamzami, N., et al., J Exp Med., 1995, 118, 1661–1672. Dead cells (DiOC₆⁻, PI⁺) took in PI after their outer cell membrane deteriorated. Dyeabsorption and the percentages of each cell type were then determined ona flow cytometer. (FIG. 16) The population of viable cells was used toestimate the effective concentration of each compound needed to causeapoptosis in 50% of CLL cells (EC₅₀). This experiment was then repeatedwith normal peripheral blood lymphocyte (PBL) cells to estimate thelethal concentration of each compound needed to kill 50% of normal cells(LC₅₀). Four drug levels (100, 250, 500, and 750 μM) were tested. Thus,most of the EC₅₀'s and LC₅₀'s were expressed as a range.

In each well of a 24-well plate, test compounds were added to 5×10⁶primary CLL or PBL cells suspended in 2 mL of RPMI-1620 medium (IrvineScientific, Santa Ana, Calif.) containing 10% fetal bovine serum (FBS),100 μg/mL penicillin, and 100 μg/mL streptomycin to get finalconcentrations of 100, 250, 500, and 750 μM. Cells alone and R-etodolacserved as controls. The plate was then incubated under a 5% CO₂atmosphere at 37° C. for 48 hours, after which 450 μL of each well wasremoved, incubated for 30 minutes with 50 μL of phosphate buffersolution (PBS) containing 40 nM DiOC₆ and 5 μg/mL PI, and analyzed byflow cytometry using a FACS caliber (Beckton-Dickinson, San Jose,Calif.). Viable cells had high DiOC₆ and low PI signal. Early apoptoticcells had low signals of DiOC₆ and PI. Dead cells had low DiOC₆ and highPI signal. The results are illustrated in Table 2 and FIG. 16.

MTT Cytotoxicity Studies.

To get a more concise measure of the EC₅₀ and confirm the flow cytometryresults, a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tertrazoliumbromide) assay using a procedure similar to that described by Mosmann,T., J Immunol Methods. 1983, 65, 55–63, was performed on all analogs.This method provided a larger number of datapoints so that a value forthe EC₅₀ could be extrapolated.

This assay was based on a procedure described by Mosmann. Briefly, ineach well of a 96-well plate, 5×10⁵ CLL cells were suspended in 100 μLof RPMI-1620 medium containing 10% FBS, 100 μg/mL penicillin, and 100μg/mL streptomycin. Serial dilution of test compounds were thenperformed in duplicate across the plate to establish a concentrationgradient from 0 to 1000 μM. After 72 hours of incubation at 37° C., thecells were incubated for 6 more hours in the presence of 20 μL of 5mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tertrazoliumbromide) before dissolving the resulting crystals in 50 μL lysis buffer15 mM HCl) overnight. Afterwards, the absorbance of each well was at 570and 650 nM in a Finstrument Microplate Reader, and the data was yGraphpad Prism Software, version 2.0b. The results are illustrated inTable 2.

TABLE 2 EC₅₀ and LC₅₀ of the etodolac derivatives in CLL cells.

MTT^(a) Flow Cytometry Compound R EC₅₀ (μM) EC₅₀ (μM) LC₅₀ (μM)R-etodolac COOH 247.4 100–250 ~500 1 CH₂OH 177.5 100–250 ~500 2CH₂OCH₃ >1000 250–500 >750 3 CH₂F 767.3 250–500 <100 4 CH₂Cl 572.2 <100<100 5 CH₃ >1000 250–500 nd 6 CONH₂ 257.9 100–250 >500 7 CH═NH 714.1250–500 250–500 8 CHO 111.3 <100 <100 9 CH₂OCH₂SCH₃ 324.5 ~250 nd 10 CH₂OAc 686.3 250–500 100–250 11  CH(OH)₂ 415.8 250–500 nd 12  CHOHCH₃272.4 100–250 250–500 13  COCH₃ >1000 250–500 >750 nd = not determined^(a)Assays were performed in duplicate.Activity Screening Assays.

Normal prostate cells (PREC, Cambrex East Rutherford N.J.), prostatecancer cell line (LnCAP, ATCC, Manassas, Va., USA), myeloma cell line(RPMI-8226, ATCC, Manassas, Va., USA), PBL (peripheral bloodleukocytes-buffy coat San Diego Blood Bank), and primary CLL cells wereincubated for one to two days in RPMI-1640 and 10% FBS (fetal bovineserum). They were plated in 96-well plates at 100,000 cells/well.Titrated concentrations of the compound to be tested were added to theculture medium. The cells were incubated three days at 37° C., 5% CO₂.Viability of the cells was assayed by standard MTT assay. Each drugconcentration was done in duplicate. The results are illustrated inTable 3.

TABLE 3 CLL (μM) LnCap RPMI-8226 Compound Structure MW by MTT (μM) (μM)(R-Etodolac)

287.36 174.1 (1st)263.1 (2nd) 95 (1st)132.0 (2nd)140 (3rd)108.4 (4th)250 (1st)197.3 (2nd)250 (3rd)139.4 (4th) 1

273.37 177.4 39.8 134.7 2

287.40 >1000 473.5 3

275.37 767.3 ~210 ~190 4

291.82 572.2 ~58 ~105 5

257.38 >1000 340 ~180 6

286.37 105200190160160 59.6 109.6 7

270.37 714.3 ~100 ~270 8

271.36 111.3 57 ~70 uM 9

333.49 324.5 ~140 121.5 10 

315.41 686.3 11 

289.38 415.8 200.9 304.9 12 

287.40 272.4 62.7 ~110 13 

285.39 686.3 39.8 53.3COX-1/COX-2 Enzyme Assays.

Compounds active in both the flow cytometry and MTT experiments werethen tested for COX inhibition at 50 μM in an enzyme assay using COX-1isolated from human platelets and human recombinant COX-2 purified frominsect cells (Spodoptera frugiperda), as described by Riendeau et. al.,Br J Pharmacol. 1997, 121, 105–117; Riendeau et. al., Can J PhysiolPharmacol. 1997, 75, 1088–1095; and Warner et. al. Proc Natl Acad. Sci.1999, 96, 7563–7568. These assays were performed by MDS Pharma Services,Bothell, Wash. The compounds were incubated in duplicate with COX-1isolated from human platelets and human recombinant COX-2 purified frominsect cells (Spodoptera frugiperda). Enzyme immunoassay quantificationof prostaglandin E₂ production from arachidonic acid in the presence (50μM) and absence of test compounds was used to determine the percentageof COX inhibition. The results are illustrated in Table 4.

TABLE 4 COX Inhibition of Compound 1 and 6. % Inhibitions^(a) Compound RCOX-1 COX-2 1 CH₂OH 0 0 6 CONH₂ 36 69 ^(a)Percentages of inhibition ofCOX-1 isolated from human platelets and purified human recombinantCOX-2. Compounds were tested at 50 μM. Assays were performed induplicate.

All of the publications and patent documents cited hereinabove areincorporated by reference herein. The invention has been described withreference to various specific and preferred embodiments and techniques.However, it should be understood that many variations and modificationsmay be made while remaining within the spirit and scope of theinvention.

1. A compound of formula (I):

wherein R¹ is lower alkyl, lower alkenyl, lower alkynyl, phenyl, benzylor 2-thienyl, R², R³, R⁴ and R⁵ are the same or different and are eachhydrogen or lower alkyl; each R⁶ is independently hydrogen, lower alkyl,hydroxy, (hydroxy)lower alkyl, lower alkoxy, benzyloxy, loweralkanoyloxy, nitro or halo, n is 1–3, R⁷ is hydrogen, lower alkyl orlower alkenyl; X is oxy or thio, Y is (CH₂)₁₋₃, (CH₃)₁₋₃CO or(CH₂)₁₋₃SO₂; Z is (ω-(4-pyridyl)(C₂–C₄alkoxy), (ω-((R⁸)(R⁹) amino)(C₂–C₄alkoxy), an amino acid ester of (ω-(HO)(C₂–C₄))alkoxy, N(R⁸)CH(R⁸)CO₂H,1′-D-glucuronyloxy, OH, (C₂–C₄)acyloxy, SO₃H, PO₄H₂, N(NO)(OH), SO₂NH₂,PO(OH)(NH₂), OCH₂CH₂N(CH₃)₃ ⁺, amino, lower alkylamino, di(loweralkyl)amino, phenylamino, or tetrazolyl; wherein R⁸ and R⁹ are each H,(C₁–C₃)alkyl or together with N, are a 5- or 6-membered heterocyclicring having 1–3 N(R⁸), S or nonperoxide O; or Y-Z is (CH₂)₁₋₃R¹⁰ whereinR¹⁰ is OH, (C₂–C₄)acyloxy, SO₃H, PO₄H₂, N(NO)(OH), SO₂NH₂, PO(OH)NH_(2,)provided that when n is 1, R⁶ is hydrogen, and R¹ is methyl, then —Y-Zis not —CH₂CH₂—OH, —CH₂—OH, —CH₂CH₂—OC(O)CH₃, or —CH₂—OC(O)CH₃; ortetrazolyl; and when n is 1, R⁶ is 8-ethyl, and R¹ is ethyl, then —Y-Zis not —CH₂CH₂—OH, or —CH₂CH₂—OC(O)CH_(3;) or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1 wherein Z is theL-valine or L-glycine ester of a 2-hydroxyethoxy group.
 3. A compound offommia (I):

wherein R¹ is lower alkyl, lower alkenyl, lower alkynyl, phenyl, benzyl,or 2-thienyl, R², R³, R⁴ and R⁵ are the same or different and are eachhydrogen or lower alkyl; each R⁶ is independently hydrogen, lower alkyl,hydroxy, (hydroxy)lower alkyl, lower alkoxy, benzyloxy, loweralkanoyloxy, nitro or halo; n is 1–3; R⁷ is hydrogen, lower alkyl orlower alkenyl; X is oxy or thio, Y is —CH₂—and Z is —CH₂OH, —CONH₂,—CHOHCH₃, —CH₂OCH₂SCH₃, or —CH(OH)₂; provided that when n is 1, R⁶ ishydrogen, and R¹ is methyl, then —Y-Z is not —CH₂CH₂—OH; and when n is1, R⁶ is 8-ethyl, and R¹ is ethyl, then —Y-Z is not —CH₂CH₂—OH; or apharmaceutically acceptable salt thereof.
 4. The compound of claim 1wherein Z is N-morpholinoethoxy.
 5. The compound of claim 1 wherein eachR⁸ is H, CH₃ or i-Pr.
 6. The compound of claim 1 wherein Z isOCH₂CH₂N(CH₃)₃ ⁺.
 7. A composition comprising the compound of claim 1 incombination with a pharmaceutically acceptable carrier.
 8. Thecomposition of claim 7 which is a tablet, granule or capsule.
 9. Thecomposition of claim 7 wherein the carrier is an aqueous vehicle. 10.The composition of claim 9 which is an aqueous solution.
 11. Thecomposition of claim 7 wherein the tablet is an enterically coateddosage form.